PROJECT SUMMARY Development of a novel exosome-based enzyme replacement therapy for the treatment of human lysosomal storage diseases Intracellular delivery of protein therapeutics has transformative impact on the treatment of devastating human diseases like cancer, infection and hereditary disorders. Lysosomal storage diseases (LSDs) are the largest group of hereditary metabolic disorders caused by a deficiency of lysosomal enzymes. Current standard of treatment for LSDs is enzyme replacement therapy (ERT) using recombinant enzymes. However, ERT is only available to 6 out of 50 LSDs and the recombinant enzymes are extremely expensive ($200,000/year for the life) and only produce limited benefits in mild to moderate patients. Moreover, ERT has no effects in severe cases with vital organ involvements such as brain, heart and lung, presumably attributable to the poor bioavailability of these tissues to recombinant enzymes. Exosomes are cell-derived nano-vesicles that play an important role in mediating cell-to-cell communication. As nano-carriers, exosomes can shuttle a large amount of macromolecules between various tissues and organs. Because their intrinsic tissue-penetrating ability, exosomes represent a new and promising class of nanomedicine for intracellular targets. However, the lack of engineering strategy to load protein therapeutics onto exosomes has become a major hurdle for exosome-based nano- medicine. We aim to develop a novel genetic approach for producing enzyme-loaded exosomes for the potential treatment of LSDs. For proof in concept, we choose Gaucher disease, one of the most common types of LSDs, as our study model. In this proposed study we will: (1) establish genetic strategies for loading ?-glucocerebrosidase (GBA) enzymes via anchoring vesicular stomatitis virus glycoprotein (VSVG) onto exosomes in human producing cells, and determine molecular pathways of exosome-targeting and therapeutic enzyme loading onto exosomes using fluorescence monitoring and confocal microscope techniques, (2) establish procedures of exosome purification, and characterize the physical and biochemical properties of the isolated exosomes as well as their ability for targeted delivery of loaded lysosomal enzymes to recipient human cells, (3) establish GBA knockout human cell model and employ it to determine the safety and efficacy of enzyme replacement with enzyme-loaded exosomes. Successful completion of this study will build a novel and general platform of ERT for the potential treatment of Gaucher disease and other LSDs with neurological complications.